Bacterial collagenase gene of Vibrio alginolyticus
A collagenase gene derived from bacteria of the species Vibrio alginolyticus is disclosed. A recombinant vector containing the gene, a host cell transformed with a plasmid containing the gene and a process for the production of a collagenase by using the host cells are also disclosed.
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The present invention relates to a collagenase gene derived from Vibrio alginolytic, a recombinant vector integrating the gene, a host cell transformed with the vector, and the use thereof.
BACKGROUND OF THE INVENTIONCollagen which constitutes connective tissues of animals is composed of three polypeptide chains, each basic unit of which has a molecular weight of about 95,000. These polypeptide chains form a counterclockwise triplex spiral structure. The amino acid sequence of each polypeptide chain of the collagen molecule is a repetition of Gly-Pro-X-Gly (wherein the three-letter code representing amino acid residues SEQ ID No: 1) used herein are those according to IUPAC-IUB standards and X represents various amino acid residues) and the polypeptides are intramolecularly or intermolecularly cross-linked. This specific spiral structure of collagen brings about tough mechanical properties and chemical stability and, therefore, collagen resists degradation by ordinary proteases and only a collagenase can degrade collagen.
A collagenase does not act on ordinary proteins, but acts on only the above collagen or its modified product, gelatin. Collagenases are produced by microorganisms and, among these collagenases, the study on the collagenase known as Achromobacter collagenase which is derived from Vibrio alginolyticus chemovar. iophagus is most advanced. It has been known that this collagenase has a higher specific activity in comparison with collagenases derived from other sources [V. Keil-Dlouha and B. Keil, Biochim. Biophys. Acta, 522, 218-228 (1978)]. Achromobacter collagenase has a molecular weight of 110,000 and is stable at pH 6 to 7. The optimum pH is about pH 7.4. The collagenase, which is inactivated by EDTA and o-phenanthroline [V. Keil-Dlouha, Biochim. Biophys. Acta, 429, 239-251 (1976)], is a metalloprotease containing zinc, and breaks the synthetic substrate, PZ-Pro-Leu-Gly-Pro-D-Arg, between Leu and Gly [B. Keil, A. M. Gilles, A. Lecroisey, N. Hurion and N. T. tong, FEBS Lett., 56, 292-296 (1975); A. Lecroisey, V. Keil-Dlouha, D. R. Woods, D. Perrin and B. Keil, FEBS Lett., 59, 167-172 (1975); N. T. Tong, A. Tsugita and V. Keil-Dlouha, Biochim. Biophys. Acta, 874, 296-304 (1986)].
In view of the specific property of a collagenase, various uses have been expected and realized. For example, a collagenase is used for treatment of various injuries of any substrate having a structure rich in collagen. Examples of such injuries include burn, ulcer, scab, white hard scab of collagen base, cheloid, necrosis, particularly, necrosis by decubitus or ulcer, and the like.
A collagenase is also used for treatment of dental caries. Namely, the dental pulp is mainly composed of a dense calcareous material and collagen. In the case of dental caries, a tooth is cracked or a hole is made and calcium is leaked therefrom. Accordingly, the calcareous material is lost, and the remaining frame becomes porous and is liable to be a hotbed of bacterial infection. However, since a collagenase dissolves the porous collagen, the hotbed can be removed by washing with water. A collagenase does not act on healthy calcareous collagen.
In addition, a collagenase can be used as an agent for making meat tender. Toughness of meat is mainly caused by tendon, the main component of which is collagen. Proteases such as papain and the like are used to make meat tender by degrading tendon. However, collagen is hardly degraded by ordinary proteases. On the other hand, non-specific proteases such as papain also degrade proteins such as actin, myosin and the like which have great influence on the texture 0f meat. Therefore, the texture of meat is destroyed by treatment with papain. In this respect, since a collagenase degrades only collagen which causes toughness of meat, but does not degrade other proteins which have great influence on the texture of meat, the enzyme is a protease most suitable for an agent for making meat tender.
In the use of a collagenase for the above purposes, there is a problem that it is very difficult to obtain a collagenase at a low cost. Namely, in order to obtain Achromobacter collagenase, its producer, Vibrio alginolyticus, is cultivated and the collagenase is recovered from the culture solution and purified. However, in this respect, there is a problem that the yield of collagenase by the producer is very low such as 10 mg/liter. Further, there is another problem that any collagenase is not produced by the producer unless a certain specific inducing substance is added to a culture medium. Thus, it is very difficult to obtain the collagenase in a large amount at a low cost.
Although it is possible to employ genetic engineering techniques to solve these problems, no gene of Achromobacter collagenase is yet available. Therefore, no genetic engineering technique can be employed to produce the enzyme in a large amount.
OBJECTS OF THE INVENTIONThe present inventors have studied intensively to solve these problems. As a result, the present inventors have successfully obtained a gene of Achromobacter collagenase and clarified its amino acid sequence, whereby it is possible to produce Achromobacter collagenase in a large amount in a suitable host and to improve the availability of a collagenase by means of genetic engineering techniques.
One object of the present invention is to provide a gene encoding Achromobacter collagenase.
Another object of the present invention is to provide a recombinant vector containing the gene of Achromobacter collagenase.
Still another object of the present invention is to provide a host cell transformed by the vector.
Still another object of the present invention is to provide a process for the production of Achromobacter collagenase by using the host cell.
These objects as well as other objects and advantages of the present invention will become apparent to those skilled in the art from the following description with reference to the accompanying drawings.
BRIEF EXPLANATION OF DRAWINGSFIG. 1 is a restriction map of a DNA fragment of 7.0 kb containing the collagenase gene of the present invention which composes the plasmid pLCO-1. In FIG. 1, the arrow at the bottom part represents the collagenase structural gene region as well as the direction of transcription. The number in the parentheses is that of the base. The dotted line means that the restriction cleavage site can not be specified between the two sites.
FIG. 2 is the entire DNA base sequence of a DNA fragment containing the collagenase gene and an amino acid sequence of the collagenase deduced from the base sequence (SEQ ID No: 2). In FIG. 2, the regions underlined by the solid line represent the parts corresponding to the partial amino acid sequences (see Example 3 hereinafter) of the purified collagenase.
FIG. 3 illustrates an analytical result of Western blotting of a collagenase gene product in Escherichia coli. In FIG. 3, the arrows represent the migration positions of markers having various molecular weights which were subjected to Western blotting simultaneously.
SUMMARY OF THE INVENTIONAccording to the present invention, there is provided a collagenase gene derived from bacteria of the species Vibrio alginolyticus.
The present invention also provides a recombinant vector containing the above gene of the present invention or a biologically equivalent thereof, and a host cell transformed with a plasmid containing the gene of the present invention.
The present invention further provides a process for the production of a collagenase which comprises cultivating the host cells to produce the collagenase and recovering the collagenase thus produced from the cells or culture solution.
DETAILED DESCRIPTION OF THE INVENTIONThe bacteria of the species Vibrio alginolyticus to be used for obtaining the collagenase gene of the present invention is not specifically limited and any known producer of Achromobacter collagenase can be used. For example, Vibrio alginolyticus disclosed by I. Emonto et al. in Int. J. Syst. Bacteriol., 33,451-459, 1983. Further, the isolation of a bacterial DNA, preparation of a gene library and screening can be conducted according to the conventional methods as shown in Examples hereinafter.
As host cells, Escherichia coli, Bacillus subtilis and the like can be used. As vectors, pUC18, pUC19, pBR322, pGEM3, pGEM4 and the like which can be replicated in Escherichia coli as well as pUBl10, pE194, pC194 and the like which can be replicated in Bacillus subtilis can be used.
In order to produce a collagenase by using the host cells transformed by the plasmid containing the collagenase gene thus obtained, for example, the cells are cultivated in a suitable culture medium containing suitable carbon sources, nitrogen sources and trace amounts of metallic elements according to the method described by A. Lecroisey et al. in FEBS Lett., 59,167-172, 1975. The resultant culture is recovered by the conventional method and the supernatant of the culture is subjected to ammonium sulfate precipitation (60% saturated). Then, the enzyme is purified by chromatography, for example, DEAE column chromatography, Sephadex G-100 column chromatography and the like to obtain the desired collagenase.
The collagenase thus obtained can be used according to the same manner as that of known collagenases.
The following Examples further illustrate the present invention in detail but are not to be construed to limit the scope thereof.
Example 1Preparation of gene library
According to the conventional method [e.g., Saito-Miura Method (H. Saito and K. Miura, Biochem. Biophys. Acta, 72, 619, 1963), etc.], chromosomal DNA was isolated from an Achromobacter collagenase producer, Vibrio alginolyticus obtained from The National Collection of Industrial Bacteria (NCIB) [VIBRIO ALGINOLYTICUS SUBSP, IOPHAGUS AL, 11038 R. L. Welton/South African cured hides/(62SC)]. The DNA was partially cleaved with the restriction enzyme Sau3A1 and fractionated by agarose gel electrophoresis to obtain a DNA fragment of 5 kb or more. The DNA fragment was ligated to the vector pUC18 treated with BamHl by T4 ligase. Escherichia coli JM101 was transformed by the resultant ligation mixture according to the conventional method (e.g., M. Mandel and A. Higa, J. Mol. Biol., 53, 154, 1970) to obtain a gene library of Vibrio alginolyticus as an ampicillin resistant transformant.
Example 2Screening of gene library
In order to select a transformant producing the collagenase, Achromobacter collagenase antibody was prepared according to the conventional method [e.g., Zoku-Seikagaku Zikken Ho (Methods of Biochemical Experiments Second Series), edited by the Biochemical Society of Japan, Vol. 5, pp. 1 to 25, 1986]. Namely, a purified collagenase (1 mg) was mixed with Freund's incomplete adjuvant and a rabbit was immunized by subcutaneously injecting the mixture. Further, the same operation was repeated once a week for 3 weeks to give booster immunization. In the fourth week, whole blood was collected and an IgG fraction was prepared by ammonium sulfate fractionation. The antibody was labeled with peroxidase according to a known method such as that using sodium periodate [Meneki Zikkensosa Ho (Methods for Immunological Experiments) VI, edited by the Immunological Society of Japan, p 1835].
The anti-collagenase antibody labeled with the enzyme thus Obtained was used for selection of clones expressing an antigen which was able to react with the antibody from the above-prepared gene library to obtain plasmids pLCO-1, pLCO-2 and pLCO-3.
The plasmid pLCO-1 has a DNA fragment of about 7.0 kb derived from Vibrio alginolyticus inserted therein. The restriction map of the DNA fragment inserted in pLCO-1 is shown in FIG. 1.
Further, Escherichia coli JM101 containing the plasmid pLCO-1 was named as Escherichia coli SAM 1514 and deposited with Fermentation Research Institute, Agency of Industrial Science and Technology (FRI) under Budapest treaty on Nov. 22, 1989 under the accession number of FERM BP-3113.
Example 3Determination of amino acid sequence
Partial amino acid sequences of Achromobacter collagenase were determined as follows:
Purified Achromobacter collagenase was partially hydrolyzed with trypsin or protease V8, respectively according to the conventional method (e.g., Zoku-Seikagaku Zikken Ho, Vol. 2, pp. 260-270, edited by the Biochemical Society of Japan). The peptide fragments thus obtained were purified by high performance liquid chromatography and then their amino acid sequences were determined by automatic Edman degradation method.
As a result, it has been found that Achromobacter collagenase of the present invention has the amino acid sequences of the following 20 peptide fragments.
(a): S Q L S R
(b): I Y R
(c): Y T G N A S S V V K
(d): A S S I G A E D E F M A A N A G R E
(e): E S V D A F V N
(f): Q G N W I N Y K
(g): M G Y E E G Y F H Q S L
(h): A L G D F A L R
(i): W G Y L A V R
(j): A G Y Y A E
(k): V W W S E
(l): W V T P A V K E
(m): L D G R F D L Y G G F S H P T E K
(n): Y N D N I S F
(o): S S T D Y G K Y A G P I F D
(p): G D P S Q P G N I P N F I A Y E
(q): Y V H Y L D G R F D
(r): T A S Y Y A D C S E
(s): W N D Q Y
(t): G Y T G G G S D E L
wherein A is alanine, C is cysteine, D is aspartic acid, E is glutamic acid, F is phenylalanine, G is glycine, H is histidine, I is isoleucine, K is lysine, L is leucine, M is methionine, N is asparagine, P is proline, Q is glutamine, R is arginine, S is serine, T is threonine, V is valine, W is tryptophan and Y is tyrosine.
Example 4Determination of DNA base sequence
The base sequence of the fragment of 4.1 kb in the DNA fragment of 7.0 kb composing the plasmid pLCO-1 was determined as follows:
Namely, the plasmid pLCO-1 was cleaved with various restriction enzymes to prepare DNA fragments of about 500 bp. These fragments were cloned into phage M13 and the DNA base sequences of each recombinant phage DNA were determined by dideoxy method (F. Sanger et al., Proc. Nat. Acad. Sci. USA, 74, 5963-5967, 1977). A DNA base sequence of about 4 kb was determined by joining respective sequences of DNA fragments.
The DNA base sequence thus determined is shown in FIG. 2. The DNA base sequence is composed of 4054 base pairs and the entire region of Achromobacter collagenase gene is contained therein. As seen from FIG. 2, there is an open reading frame corresponding to the collagenase which is composed of 2442 base pairs initiated from ATG of Base Nos. 1337 to 1339 and terminated by TAG of Base Nos. 3779 to 3781 in the DNA sequence. The ribosome binding site, GAAGAAA, is located at 5 bp prior to the ATG initiation codon.
When amino acid sequences deduced from the DNA base sequence thus determined were compared with the partial amino acid sequences determined in Example 3, the following 20 amino acid sequences agreed with each other. ##STR1##
Example 5Analysis of gene product
A recombinant plasmid for the mass production of the collagenase gene in Escherichia coli was prepared.
BamHI linker was inserted into HpaI site at Base No. 1213 on the DNA fragment of 7 kb composing the plasmid pLCO-1 and SalI linker was inserted into EcoRV site at Base NO. 3936 on the DNA fragment. The resultant pLCO-1 containing these two linkers was cleaved with BamHI and SalI to obtain a DNA fragment of 2.7 kb containing the entire collagenase gene. The DNA fragment was recovered and inserted into BamHI/SalI site of the vector pUC18 to obtain a recombinant plasmid pHUC14. Escherichia coli JM109 was transformed with the recombinant plasmid pHUC14 to obtained a recombinant Escherichia coli for the mass production of the collagenase.
The Achromobacter collagenase gene product in the recombinant Escherichia coli was analyzed by electrophoresis and Western blotting. Western blotting was conducted by modified Burnette method [Burnette, W. N., Anal. Biochem., 112,680-685 (1981)].
The recombinant Escherichia coli containing the collagenase gene was cultured in L-broth containing 1 mM of IPTG at 37.degree. C. for 17 hours. The cells were collected by centrifugation and broken by sonication. The sonicated cell suspension was fractionated by SDS-polyacrylamide gel electrophoresis. The protein thus fractionated was translated to a nitrocellulose membrane by Western blotting and color of only the bands of the collagenase was developed by an anti-collagenase antibody from a rabbit and an anti-rabbit IgG antibody labeled with peroxidase according to the same manner as that described above. As shown in FIG. 3, many bands which reacted with the anti-collagenase antibody mainly composed of protein having the molecular weight of about 85 kd were observed in Escherichia coli JM109 containing pHUC14. In Escherichia coli JM109 containing pLCO-1, protein which reacted with the anti-collagenase antibody was also observed, although the amount thereof was very small. On the other hand, in Escherichia coli JM109 containing no recombinant plasmid used as a control, no protein which reacted with the anti-collagenase antibody was observed.
These results show that protein which is an immunologically equivalent to Achromobacter collagenase is produced in Escherichia coli containing the recombinant plasmid pLCO-1 or pHUC14 and a large amount of the protein is produced by Escherichia coli containing the recombinant plasmid pHUC14.
Example 6Collagenase activity of transformant
Collagenase activity of Escherichia coli containing Achromobacter collagenase gene was measured by using the synthetic substrate, 4-phenylazo-benzyloxycarbonyl-L-Pro-Leu-Gly-L-Pro-D-Arg.HCl (PZ-PLGPR).
The measurement of collagenase activity and the definition of the unit of the activity (U) are disclosed in International Publication WO 84/02653.
The sonicated cell solution was prepared according to the same manner as that disclosed in Example 5. As shown in Table 1, an coliagenase activity was observed in Escherichia coli JM109 containing the plasmid pHUC14. No activity was observed in Escherichia coli JM109 containing the plasmid pLCO-1 or containing no plasmid.
In view of these results, it is clear that the gene product in the recombinant Escherichia coli containing the collagenase gene has collagenase activity. Although no collagenase activity is observed in Escherichia coli containing the plasmid pLCO-1, this would be due to a low expression level.
TABLE 1
______________________________________
Collagenase activity of recombinant Escherichia coli
Plasmid Collagenase activity
______________________________________
no <5
pLCO-1 <5
pUC14 189
______________________________________
Note) "Plasmid" means Escherichia coli JM109 containing the corresponding
plasmid.
__________________________________________________________________________
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 25
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GlyProXaaGly
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4054 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: genomic DNA
(iii ) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Vibrio alginolyticus
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
( I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION: /note="location 1337 to 3781 base
pairs open reading frame"
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GATCGTACCAGTCATTATATCTGCTGCTGCGATGTACTTTTTCTACACACGCCTTGGCTT60
ATCACAAACTTATCTAGGCGTCATTTTGGCACACGCTGCGTTAGGTACGCCTTTTGTC GT120
CATTACCGTTACTGCGACGTTAAGTGGCTTTGACCATAGCTTGGTAAAAGCGGCGGCTAG180
CTTAGGAGCAAACCCTGTTTATACTTTCAGACACATTACCTTTAAGCTGATTCGTCCGGG240
GATGATTTCTGGCGGCTTGTTTGCCTTTGAGC ATCGTTCGACGAGGTTGTGGTGGCGTTA300
TTCCTGACTGGGGCAGAACAAAAAACCGTTCCGAGGCAGATGTGGTCAGGAATTCGAGAG360
CAAATTAGTCCGACCATATTGGCGGTCGCTACGTTGTTGATTTTTATGTCGGTGTGTTTG420
CTCGTGA CGTTAGAAGTTTTGCGTAGACGTAATATACGCATTCGAGGCATTCAAGAATAA480
CCAGACTTTTTCTTTGTTGGTCACTATGCACTTTTGTTTAGGGGCACCTCAATTTTTGAC540
CAAAGGCGCCTTATTGTAGGCGCTTTTCTTTTGTGTTTGTCGTCAGCGAT GACTGACATG600
TCACTCTTGTAGCTTAATGCCAGCCTGCTCGAAACGTGCCAAAGGCGACTTGTTCGATTG660
CTGATACCAACTAGCTGGCATGTTTGGTTTAAGTCGGGCTTTTTCATCCTTGCTAATCAC720
AACATAGTGTAAAAATGCATCAAGT GTGGTTCCCATCACGTCAAACGTCATTTCCCCACC780
TTTGCTGATGGTTTTCAATAATCCAACGACGGCTTCCTCAGGGACTAACGGTTCAACCAC840
CGCGTTTTTTCGCTCTTTTATTGATGTTGAAGCGGGCAGGTTTGCACGTGCCGTCTCCCA900
GCAAACTTTGTTGGTTTTACTGACAAGGTTGTGCTTGTAGCAGTAGTAACTGAATAAAAA960
GCGTGGCAGATTTTGCCAGCGTTCGGCTTGGACTTCTAACCAAAGGTGTTGTAGCTCAAT1020
ATCAGCGTCGGTCATAGGAGTTGTTTAGCAAAAAGAAAAGAAA CCATTTTATCGCTTTTG1080
TGAGGAGCAATAAAAGATATTTGAATGGAAAGATAAACAACTAGTTTATCAATATTACTA1140
AAGCAAATAGATTTCTGGCAAGCCCGTGCAACGCAACTCGAGTACCAAAAACTGATACCG1200
CCACATTCGGTTGTTAAC AAAATGTTTCTTCTTGTCTTGCGAGTAGATTATATGGAGATG1260
CTCTTGCAGTAATAAGGGCAGTGGCGATGCAAAAGACGTAATGCATCTAAGGAAAACTCA1320
ATATAGAAGAAATTAGATGGAACTGAAGATTTTGAGTGTCGCGATTGCGACA 1372
MetGluLeuLysIleLeuSerValAlaIleAlaThr
1510
ACATTAACCAGCACTGGCGTATTTGCGTTAAGCGAGCCAGTTTCTCAA 1420
ThrLeuThrSerThrGlyValPheAlaLeuSerGluProValSerGln
152025
GTTACAGAGCAACATGCACATTCGGCTCATACACACGGTGTTGAATTC 1468
ValThrGluGlnHisAlaHisSerAlaHisThrHisGlyValGluPhe
303540
AATCGAGTTGAATACCAACCAACCGCAACTCTCCCAATTCAGCCCTCT1516
A snArgValGluTyrGlnProThrAlaThrLeuProIleGlnProSer
45505560
AAGGCAACTCGAGTACAGTCACTTGAAAGCCTTGATGAGTCGAGCACT 1564
LysAlaThrArgValGlnSerLeuGluSerLeuAspGluSerSerThr
657075
GCTTGTGATTTGGAGGCATTGGTTACCGAAAGCAGTAACCAATTGATC 1612
AlaCysAspLeuGluAlaLeuValThrGluSerSerAsnGlnLeuIle
808590
AGCGAAATTTTAAGTCAGGGCGCGACGTGTGTGAACCAGTTATTCTCT 1660
SerGluIleLeuSerGlnGlyAlaThrCysValAsnGlnLeuPheSer
95100105
GCTGAAAGTCGGATTCAAGAGTCGGTATTTAGCTCCGATCATATGTAC 1708
AlaGluSerArgIleGlnGluSerValPheSerSerAspHisMetTyr
110115120
AACATCGCTAAGCACACTACGACGTTGGCGAAGGGGTATACGGGTGGC1756
A snIleAlaLysHisThrThrThrLeuAlaLysGlyTyrThrGlyGly
125130135140
GGGAGCGATGAACTAGAAACGTTGTTCTTATACTTACGCGCGGGTTAT 1804
GlySerAspGluLeuGluThrLeuPheLeuTyrLeuArgAlaGlyTyr
145150155
TACGCCGAGTTTTACAATGACAACATCTCATTTATTGAATGGGTCACC 1852
TyrAlaGluPheTyrAsnAspAsnIleSerPheIleGluTrpValThr
160165170
CCAGCGGTGAAAGAATCAGTGGATGCGTTTGTTAACACAGCAAGCTTC 1900
ProAlaValLysGluSerValAspAlaPheValAsnThrAlaSerPhe
175180185
TACGAGAACAGCGACCGTCACGGCAAAGTGCTTAGTGAGGTCATCATC 1948
TyrGluAsnSerAspArgHisGlyLysValLeuSerGluValIleIle
190195200
ACTATGGATAGTGCGGGCTTGCAGCACGCGTACTTACCGCAAGTGACC1996
T hrMetAspSerAlaGlyLeuGlnHisAlaTyrLeuProGlnValThr
205210215220
CAGTGGCTTACTCGTTGGAATGATCAATACGCCCAGCACTGGTATATG 2044
GlnTrpLeuThrArgTrpAsnAspGlnTyrAlaGlnHisTrpTyrMet
225230235
CGCAATGCGGTTAACGGTGTTTTCACTATTTTGTTTGGTGGGCAGTGG 2092
ArgAsnAlaValAsnGlyValPheThrIleLeuPheGlyGlyGlnTrp
240245250
AACGAGCAATTTGTGCAAATAATTGGCAACCAAACGGACCTTGCCAAA 2140
AsnGluGlnPheValGlnIleIleGlyAsnGlnThrAspLeuAlaLys
255260265
GCTTTAGGCGATTTTGCTCTAAGGGCGTCATCAATCGGTGCTGAAGAT 2188
AlaLeuGlyAspPheAlaLeuArgAlaSerSerIleGlyAlaGluAsp
270275280
GAGTTTATGGCCGCGAATGCGGGGCGAGAGCTCGGGCGTCTGACCAAG2236
G luPheMetAlaAlaAsnAlaGlyArgGluLeuGlyArgLeuThrLys
285290295300
TATACGGGTAACGCGAGTTCTGTTGTGAAGAGTCAGCTGAGTCGAATC 2284
TyrThrGlyAsnAlaSerSerValValLysSerGlnLeuSerArgIle
305310315
TTTGAACAGTATGAAATGTATGGTCGGGGTGACGCGGTTTGGCTTGCG 2332
PheGluGlnTyrGluMetTyrGlyArgGlyAspAlaValTrpLeuAla
320325330
GCGGCGGACACCGCCTCATATTACGCAGATTGTAGTGAGTTCGGAATT 2380
AlaAlaAspThrAlaSerTyrTyrAlaAspCysSerGluPheGlyIle
335340345
TGTAATTTCGAAACTGAGCTAAAAGGCTTGGTGCTATCGCAAACTTAT 2428
CysAsnPheGluThrGluLeuLysGlyLeuValLeuSerGlnThrTyr
350355360
ACTTGTAGCCCGACAATCCGAATTTTGTCTCAGAATATGACGCAAGAG2476
T hrCysSerProThrIleArgIleLeuSerGlnAsnMetThrGlnGlu
365370375380
CAACACGCGGCCGCATGTTCTAAAATGGGTTACGAAGAGGGTTACTTT 2524
GlnHisAlaAlaAlaCysSerLysMetGlyTyrGluGluGlyTyrPhe
385390395
CATCAGTCATTAGAAACTGGTGAACAGCCAGTAAAAGATGACCACAAT 2572
HisGlnSerLeuGluThrGlyGluGlnProValLysAspAspHisAsn
400405410
ACTCAGCTCCAAGTCAATATATTCGATTCAAGTACCGATTATGGTAAG 2620
ThrGlnLeuGlnValAsnIlePheAspSerSerThrAspTyrGlyLys
415420425
TACGCAGGGCCAATTTTCGATATTAGTACTGACAATGGCGGTATGTAC 2668
TyrAlaGlyProIlePheAspIleSerThrAspAsnGlyGlyMetTyr
430435440
TTGGAGGGCGACCCTTCCCAGCCGGGGAATATTCCCAACTTTATTGCT2716
L euGluGlyAspProSerGlnProGlyAsnIleProAsnPheIleAla
445450455460
TATGAAGCCTCTTATGCGAACGCAGATCACTTTGTCTGGAACTTAGAG 2764
TyrGluAlaSerTyrAlaAsnAlaAspHisPheValTrpAsnLeuGlu
465470475
CACGAATACGTGCATTACTTAGATGGTCGATTTGATCTCTATGGAGGG 2812
HisGluTyrValHisTyrLeuAspGlyArgPheAspLeuTyrGlyGly
480485490
TTTAGTCATCCAACTGAAAAAATAGTGTGGTGGAGTGAAGGCATTGCA 2860
PheSerHisProThrGluLysIleValTrpTrpSerGluGlyIleAla
495500505
GAGTATGTCGCTCAAGAAAATGACAACCAAGCAGCACTTGAGACGATT 2908
GluTyrValAlaGlnGluAsnAspAsnGlnAlaAlaLeuGluThrIle
510515520
CTAGACGGTTCGACATATACCTTAAGTGAGATTTTCGAGACTACTTAT2956
L euAspGlySerThrTyrThrLeuSerGluIlePheGluThrThrTyr
525530535540
GATGGGTTTGATGTCGATCGAATTTATCGTTGGGGGTACTTAGCTGTA 3004
AspGlyPheAspValAspArgIleTyrArgTrpGlyTyrLeuAlaVal
545550555
CGTTTTATGTTTGAAAATCATAAAGATGACGTAAACCAAATGCTGGTG 3052
ArgPheMetPheGluAsnHisLysAspAspValAsnGlnMetLeuVal
560565570
GAAACACGCCAAGGGAATTGGATCAATTACAAGGCCACGATCACCCAA 3100
GluThrArgGlnGlyAsnTrpIleAsnTyrLysAlaThrIleThrGln
575580585
TGGGCGAATTTGTATCAAAGTGAGTTTGAGCAGTGGCAGCAAACCCTT 3148
TrpAlaAsnLeuTyrGlnSerGluPheGluGlnTrpGlnGlnThrLeu
590595600
GTCTCAAATGGTGCTCCTAATGCAGTCATAACCGCAAACAGTAAGGGG3196
V alSerAsnGlyAlaProAsnAlaValIleThrAlaAsnSerLysGly
605610615620
AAAGTCGGTGAAAGCATTACATTTAGCAGTGAAAACAGTACAGACCCA 3244
LysValGlyGluSerIleThrPheSerSerGluAsnSerThrAspPro
625630635
AACGGGAAGATCGTCAGCGTCTTATGGGACTTCGGTGATGGCTCGACA 3292
AsnGlyLysIleValSerValLeuTrpAspPheGlyAspGlySerThr
640645650
AGTACACAAACCAAGCCGACGCACCAATATGGGAGTGAAGGGGAGTAT 3340
SerThrGlnThrLysProThrHisGlnTyrGlySerGluGlyGluTyr
655660665
TCGGTCAGCCTAAGTGTGACAGACAGTGAAGGCTTGACGGCAACCGCC 3388
SerValSerLeuSerValThrAspSerGluGlyLeuThrAlaThrAla
670675680
ACTCATACTGTTGTTATCTCAGCGTTGGGCGGTAATGACACATTGCCA3436
T hrHisThrValValIleSerAlaLeuGlyGlyAsnAspThrLeuPro
685690695700
CAAGACTGCGCGGTGCAAAGTAAAGTAAGCGGTGGGCGCTTAACAGCA 3484
GlnAspCysAlaValGlnSerLysValSerGlyGlyArgLeuThrAla
705710715
GGAGAACCAGTTTGCTTGGCAAATCAACAAACCATTTGGCTGAGCGTA 3532
GlyGluProValCysLeuAlaAsnGlnGlnThrIleTrpLeuSerVal
720725730
CCAGCGGTGAATGAGAGCTCAAACCTGGCGATAACGACGGGGAATGGT 3580
ProAlaValAsnGluSerSerAsnLeuAlaIleThrThrGlyAsnGly
735740745
ACGGGCAACCTAAAGCTTGAATACAGTAACTCTGGTTGGCCGGATGAT 3628
ThrGlyAsnLeuLysLeuGluTyrSerAsnSerGlyTrpProAspAsp
750755760
ACTAATCTTCACGGGTGGTCAGATAATATTGGTAATGGAGAGTGTATT3676
T hrAsnLeuHisGlyTrpSerAspAsnIleGlyAsnGlyGluCysIle
765770775780
ACGTTGTCAAATCAGAGTAACTACTGGGGCTACGTTAAAGTCTCTGGT 3724
ThrLeuSerAsnGlnSerAsnTyrTrpGlyTyrValLysValSerGly
785790795
GACTTTGAGAATGCCGCCATCGTCGTTGATTTTGATGCTCAGAAGTGT 3772
AspPheGluAsnAlaAlaIleValValAspPheAspAlaGlnLysCys
800805810
CGTCAGTAGGGCAATTTAACTACGTCATTTAAACTAAGTGGAGCGCCTCGCTAACA 3828
ArgGln
TCGCGGGGGCTTTTTGTTTTTACGCCGTTATCTCTATAAAAAAAACCAGCCCGAAGGCTG3888
GCAAACAAGAAGTTTGAGATGAAAATGAAAACGTTATAAAACTTGCTGATATCCTATTTC3948
TCAATAAGTTGGGTTGTGCTTTGC AGCCAGTTTTTATCTTGCGCATCAAGAAAAAGGGCT4008
AAGCGCCTGATAGACACGTGAATGGTAATGATTAAGCCAGTCTCGC4054
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
AGTCAGCTGAGTCGA15
SerGlnLeuSerArg
15
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
( A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
( D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
TATACGGGTAACGCGAGTTCTGTTGTG AAG30
TyrThrGlyAsnAlaSerSerValValLys
1510
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F ) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GCGTCATCAATCGGTGCTGAAGATGAGTTTATGGCCGCGAATGCG45
AlaSerSerThrGlyAlaGluAspGluPheMet AlaAlaAsnAla
151015
GGGCGAGAG54
GlyArgGlu
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D ) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GAATCAGTGGATGCGTTTGTTAAC24
GluSerVa lAspAlaPheValAsn
15
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CAAGGGAATTGGATCAATTACAAG24
GlnGlyAsnTrpIleAsnTyrLys
15
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G ) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
ATGGGTTACGAAGAGGGTTACTTTCATCAGTCATTA36
MetGlyTyrGluGluGlyTyrPheHis GlnSerLeu
1510
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
GCTTTAGGCGATTTTGCTCTAAGG24
AlaLeuGlyAspPheAlaLeuArg
15
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
TGGGGGTACTTAGCTGTACGT21
TrpGlyTyrLeuAlaVal Arg
15
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
GCGGGTTATTACGCCGAG18
AlaGlyTyrTyrAlaGlu
15
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
GTGTGGTGGAGTGAA15
ValTrpTrpSerGlu
1 5
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
TGGGTCACCCCAGCGGTG AAAGAA24
TrpValThrProAlaValLysGlu
15
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 51 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I ) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
TTAGATGGTCGATTTGATCTCTATGGAGGGTTTAGTCATCCAACT45
LeuAspGlyArgPheGluLeuTyrGlyGlyPheSerHisProThr
1 51015
GAAAAA51
GluLys
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
TACAATGACAACATCTCATTT21
TyrAsnGluAsnIleSerPhe
15
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
TCAAGT ACCGATTATGGTAAGTACGCAGGGCCAATTTTCGAT42
SerSerThrGluTyrGlyLysTyrAlaGlyProIlePheGlu
1510
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
GGCGACCCTTCCCAGCCGGGGAATATTCCCAACTTTATTGCTTATGAA48
GlyAspProSerG lnProGlyAsnIleProAsnPheIleAlaTyrGlu
151015
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
TACGTGCATTACTTAGATGGTCGATTTGAT30
TyrValHisTyrLeuAspGlyArgPheAsp
1 510
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B ) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
ACCGC CTCATATTACGCAGATTGTAGTGAG30
ThrAlaSerTyrTyrAlaAspCysSerGlu
1510
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
TGGAATGATCAATAC15
TrpAsnAspGlnTyr
15
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
GGGTATACGGG TGGCGGGAGCGATGAACTA30
GlyTyrThrGlyGlyGlySerAspGluLeu
1510
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 739 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
ThrAlaCysAspLeuGluAlaLeuValThrGluSerSerAsnGlnLeu
151 015
IleSerGluIleLeuSerGlnGlyAlaThrCysValAsnGlnLeuPhe
202530
SerAlaGluSerArgIleGlnGluSerValPheSerSer AspHisMet
354045
TyrAsnIleAlaLysHisThrThrThrLeuAlaLysGlyTyrThrGly
505560
GlyGlySerAsp GluLeuGluThrLeuPheLeuTyrLeuArgAlaGly
65707580
TyrTyrAlaGluPheTyrAsnAspAsnIleSerPheIleGluTrpVal
8 59095
ThrProAlaValLysGluSerValAspAlaPheValAsnThrAlaSer
100105110
PheTyrGluAsnSerAspArgHi sGlyLysValLeuSerGluValIle
115120125
IleThrMetAspSerAlaGlyLeuGlnHisAlaTyrLeuProGlnVal
130135 140
ThrGlnTrpLeuThrArgTrpAsnAspGlnTyrAlaGlnHisTrpTyr
145150155160
MetArgAsnAlaValAsnGlyValPheThrIleLeuPheGlyGly Gln
165170175
TrpAsnGluGlnPheValGlnIleIleGlyAsnGlnThrAspLeuAla
180185190
Lys AlaLeuGlyAspPheAlaLeuArgAlaSerSerIleGlyAlaGlu
195200205
AspGluPheMetAlaAlaAsnAlaGlyArgGluLeuGlyArgLeuThr
210 215220
LysTyrThrGlyAsnAlaSerSerValValLysSerGlnLeuSerArg
225230235240
IlePheGluGlnTyrGluMetTyrG lyArgGlyAspAlaValTrpLeu
245250255
AlaAlaAlaAspThrAlaSerTyrTyrAlaAspCysSerGluPheGly
260265 270
IleCysAsnPheGluThrGluLeuLysGlyLeuValLeuSerGlnThr
275280285
TyrThrCysSerProThrIleArgIleLeuSerGlnAsnMetTh rGln
290295300
GluGlnHisAlaAlaAlaCysSerLysMetGlyTyrGluGluGlyTyr
305310315320
PheHis GlnSerLeuGluThrGlyGluGlnProValLysAspAspHis
325330335
AsnThrGlnLeuGlnValAsnIlePheAspSerSerThrAspTyrGly
3 40345350
LysTyrAlaGlyProIlePheAspIleSerThrAspAsnGlyGlyMet
355360365
TyrLeuGluGlyAspProSerGlnP roGlyAsnIleProAsnPheIle
370375380
AlaTyrGluAlaSerTyrAlaAsnAlaAspHisPheValTrpAsnLeu
385390395 400
GluHisGluTyrValHisTyrLeuAspGlyArgPheAspLeuTyrGly
405410415
GlyPheSerHisProThrGluLysIleValTrpTrpSerGl uGlyIle
420425430
AlaGluTyrValAlaGlnGluAsnAspAsnGlnAlaAlaLeuGluThr
435440445
IleLeu AspGlySerThrTyrThrLeuSerGluIlePheGluThrThr
450455460
TyrAspGlyPheAspValAspArgIleTyrArgTrpGlyTyrLeuAla
465470 475480
ValArgPheMetPheGluAsnHisLysAspAspValAsnGlnMetLeu
485490495
ValGluThrArgGlnGlyAsn TrpIleAsnTyrLysAlaThrIleThr
500505510
GlnTrpAlaAsnLeuTyrGlnSerGluPheGluGlnTrpGlnGlnThr
515520 525
LeuValSerAsnGlyAlaProAsnAlaValIleThrAlaAsnSerLys
530535540
GlyLysValGlyGluSerIleThrPheSerSerGluAsnSerThrAsp
545550555560
ProAsnGlyLysIleValSerValLeuTrpAspPheGlyAspGlySer
565570575
Thr SerThrGlnThrLysProThrHisGlnTyrGlySerGluGlyGlu
580585590
TyrSerValSerLeuSerValThrAspSerGluGlyLeuThrAlaThr
595 600605
AlaThrHisThrValValIleSerAlaLeuGlyGlyAsnAspThrLeu
610615620
ProGlnAspCysAlaValGlnSerLysVal SerGlyGlyArgLeuThr
625630635640
AlaGlyGluProValCysLeuAlaAsnGlnGlnThrIleTrpLeuSer
645650 655
ValProAlaValAsnGluSerSerAsnLeuAlaIleThrThrGlyAsn
660665670
GlyThrGlyAsnLeuLysLeuGluTyrSerAsnSerGlyTrp ProAsp
675680685
AspThrAsnLeuHisGlyTrpSerAspAsnIleGlyAsnGlyGluCys
690695700
IleThrLeuSerA snGlnSerAsnTyrTrpGlyTyrValLysValSer
705710715720
GlyAspPheGluAsnAlaAlaIleValValAspPheAspAlaGlnLys
72 5730735
CysArgGln
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 814 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO:
( xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
MetGluLeuLysIleLeuSerValAlaIleAlaThrThrLeuThrSer
151015
ThrGlyValPheAlaLeuSerGluProValSerGlnValThrGlu Gln
202530
HisAlaHisSerAlaHisThrHisGlyValGluPheAsnArgValGlu
354045
TyrGlnProThr AlaThrLeuProIleGlnProSerLysAlaThrArg
505560
ValGlnSerLeuGluSerLeuAspGluSerSer
657075
ThrAla CysAspLeuGluAlaLeuValThrGluSerSerAsnGlnLeu
808590
IleSerGluIleLeuSerGlnGlyAlaThrCysValAsnGlnLeuPhe
95 100105
SerAlaGluSerArgIleGlnGluSerValPheSerSerAspHisMet
110115120
TyrAsnIleAlaLysHisThrThrTh rLeuAlaLysGlyTyrThrGly
125130135
GlyGlySerAspGluLeuGluThrLeuPheLeuTyrLeuArgAlaGly
140145150 155
TyrTyrAlaGluPheTyrAsnAspAsnIleSerPheIleGluTrpVal
160165170
ThrProAlaValLysGluSerValAspAlaPheValAsnTh rAlaSer
175180185
PheTyrGluAsnSerAspArgHisGlyLysValLeuSerGluValIle
190195200
IleThr MetAspSerAlaGlyLeuGlnHisAlaTyrLeuProGlnVal
205210215
ThrGlnTrpLeuThrArgTrpAsnAspGlnTyrAlaGlnHisTrpTyr
220225 230235
MetArgAsnAlaValAsnGlyValPheThrIleLeuPheGlyGlyGln
240245250
TyrAsnGluGlnPheValGlnI leIleGlyAsnGlnThrAspLeuAla
255260265
LysAlaLeuGlyAspPheAlaLeuArgAlaSerSerIleGlyAlaGlu
270275 280
AspGluPheMetAlaAlaAsnAlaGlyArgGluLeuGlyArgLeuThr
285290295
LysTyrThrGlyAsnAlaSerSerValValLysSerGlnLeuSerArg
300305310315
IlePheGluGlnTyrGluMetTyrGlyArgGlyAspAlaValTrpLeu
320325330
Ala AlaAlaAspThrAlaSerTyrTyrAlaAspCysSerGluPheGly
335340345
IleCysAsnPheGluThrGluLeuLysGlyLeuValLeuSerGlnThr
350 355360
TyrThrCysSerProThrIleArgIleLeuSerGlnAsnMetThrGln
365370375
GluGlnHisAlaAlaAlaCysSerLysMetG lyTyrGluGluGlyTyr
380385390395
PheHisGlnSerLeuGluThrGlyGluGlnProValLysAspAspHis
400405 410
AsnThrGlnLeuGlnValAsnIlePheAspSerSerThrAspTyrGly
415420425
LysTyrAlaGlyProIlePheAspIleSerThrAspAsnG lyGlyMet
430435440
TyrLeuGluGlyAspProSerGlnProGlyAsnIleProAsnPheIle
445450455
AlaTyrGluAla SerTyrAlaAsnAlaAspHisPheValTrpAsnLeu
460465470475
GluHisGluTyrValHisTyrLeuAspGlyArgPheAspLeuTyrGly
480485490
GlyPheSerHisProThrGluLysIleValTrpTrpSerGluGlyIle
495500505
AlaGluTyrValAlaGlnGlu AsnAspAsnGlnAlaAlaLeuGluThr
510515520
IleLeuAspGlySerThrTyrThrLeuSerGluIlePheGluThrThr
525530 535
TyrAspGlyPheAspValAspArgIleTyrArgTrpGlyTyrLeuAla
540545550555
ValArgPheMetPheGluAsnHisLysAspAspValAsnGlnM etLeu
560565570
ValGluThrArgGlnGlyAsnTrpIleAsnTyrLysAlaThrIleThr
575580585
Gln TrpAlaAsnLeuTyrGlnSerGluPheGluGlnTrpGlnGlnThr
590595600
LeuValSerAsnGlyAlaProAsnAlaValIleThrAlaAsnSerLys
605 610615
GlyLysValGlyGluSerIleThrPheSerSerGluAsnSerThrAsp
620625630635
ProAsnGlyLysIleValSerVal LeuTrpAspPheGlyAspGlySer
640645650
ThrSerThrGlnThrLysProThrHisGlnTyrGlySerGluGlyGlu
655660 665
TyrSerValSerLeuSerValThrAspSerGluGlyLeuThrAlaThr
670675680
AlaThrHisThrValValIleSerAlaLeuGlyGlyAsnAsp ThrLeu
685690695
ProGlnAspCysAlaValGlnSerLysValSerGlyGlyArgLeuThr
700705710715
AlaGl yGluProValCysLeuAlaAsnGlnGlnThrIleTrpLeuSer
720725730
ValProAlaValAsnGluSerSerAsnLeuAlaIleThrThrGlyAsn
735740745
GlyThrGlyAsnLeuLysLeuGluTyrSerAsnSerGlyTrpProAsp
750755760
AspThrAsnLeuHisGlyTrpSer AspAsnIleGlyAsnGlyGluCys
765770775
IleThrLeuSerAsnGlnSerAsnTyrTrpGlyTyrValLysValSer
780785790 795
GlyAspPheGluAsnAlaAlaIleValValAspPheAspAlaGlnLys
800805810
CysArgGln
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
( A) LENGTH: 2217 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C ) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
ACTGCTTGTGATTTGGAGGCATTGGTTACCGAAAGCAGTAACCAATTGATCAGCGAAATT60
TTAAGTCAG GGCGCGACGTGTGTGAACCAGTTATTCTCTGCTGAAAGTCGGATTCAAGAG120
TCGGTATTTAGCTCCGATCATATGTACAACATCGCTAAGCACACTACGACGTTGGCGAAG180
GGGTATACGGGTGGCGGGAGCGATGAACTAGAAACGTTGTTCTTATACTTAC GCGCGGGT240
TATTACGCCGAGTTTTACAATGACAACATCTCATTTATTGAATGGGTCACCCCAGCGGTG300
AAAGAATCAGTGGATGCGTTTGTTAACACAGCAAGCTTCTACGAGAACAGCGACCGTCAC360
GGCAAAGTGCTTAGTGAGGTCATCATC ACTATGGATAGTGCGGGCTTGCAGCACGCGTAC420
TTACCGCAAGTGACCCAGTGGCTTACTCGTTGGAATGATCAATACGCCCAGCACTGGTAT480
ATGCGCAATGCGGTTAACGGTGTTTTCACTATTTTGTTTGGTGGGCAGTGGAACGAGCAA540
T TTGTGCAAATAATTGGCAACCAAACGGACCTTGCCAAAGCTTTAGGCGATTTTGCTCTA600
AGGGCGTCATCAATCGGTGCTGAAGATGAGTTTATGGCCGCGAATGCGGGGCGAGAGCTC660
GGGCGTCTGACCAAGTATACGGGTAACGCGAGTTCTGTTGTGAAG AGTCAGCTGAGTCGA720
ATCTTTGAACAGTATGAAATGTATGGTCGGGGTGACGCGGTTTGGCTTGCGGCGGCGGAC780
ACCGCCTCATATTACGCAGATTGTAGTGAGTTCGGAATTTGTAATTTCGAAACTGAGCTA840
AAAGGCTTGGTGCTATCGCA AACTTATACTTGTAGCCCGACAATCCGAATTTTGTCTCAG900
AATATGACGCAAGAGCAACACGCGGCCGCATGTTCTAAAATGGGTTACGAAGAGGGTTAC960
TTTCATCAGTCATTAGAAACTGGTGAACAGCCAGTAAAAGATGACCACAATACTCAGCTC1 020
CAAGTCAATATATTCGATTCAAGTACCGATTATGGTAAGTACGCAGGGCCAATTTTCGAT1080
ATTAGTACTGACAATGGCGGTATGTACTTGGAGGGCGACCCTTCCCAGCCGGGGAATATT1140
CCCAACTTTATTGCTTATGAAGCCTCTTATGCGAACGC AGATCACTTTGTCTGGAACTTA1200
GAGCACGAATACGTGCATTACTTAGATGGTCGATTTGATCTCTATGGAGGGTTTAGTCAT1260
CCAACTGAAAAAATAGTGTGGTGGAGTGAAGGCATTGCAGAGTATGTCGCTCAAGAAAAT1320
GACAACCAAGCA GCACTTGAGACGATTCTAGACGGTTCGACATATACCTTAAGTGAGATT1380
TTCGAGACTACTTATGATGGGTTTGATGTCGATCGAATTTATCGTTGGGGGTACTTAGCT1440
GTACGTTTTATGTTTGAAAATCATAAAGATGACGTAAACCAAATGCTGGTGGAAAC ACGC1500
CAAGGGAATTGGATCAATTACAAGGCCACGATCACCCAATGGGCGAATTTGTATCAAAGT1560
GAGTTTGAGCAGTGGCAGCAAACCCTTGTCTCAAATGGTGCTCCTAATGCAGTCATAACC1620
GCAAACAGTAAGGGGAAAGTCGGTGAAAGC ATTACATTTAGCAGTGAAAACAGTACAGAC1680
CCAAACGGGAAGATCGTCAGCGTCTTATGGGACTTCGGTGATGGCTCGACAAGTACACAA1740
ACCAAGCCGACGCACCAATATGGGAGTGAAGGGGAGTATTCGGTCAGCCTAAGTGTGACA1800
GACAG TGAAGGCTTGACGGCAACCGCCACTCATACTGTTGTTATCTCAGCGTTGGGCGGT1860
AATGACACATTGCCACAAGACTGCGCGGTGCAAAGTAAAGTAAGCGGTGGGCGCTTAACA1920
GCAGGAGAACCAGTTTGCTTGGCAAATCAACAAACCATTTGGCTGAGCG TACCAGCGGTG1980
AATGAGAGCTCAAACCTGGCGATAACGACGGGGAATGGTACGGGCAACCTAAAGCTTGAA2040
TACAGTAACTCTGGTTGGCCGGATGATACTAATCTTCACGGGTGGTCAGATAATATTGGT2100
AATGGAGAGTGTATTACGTTGTC AAATCAGAGTAACTACTGGGGCTACGTTAAAGTCTCT2160
GGTGACTTTGAGAATGCCGCCATCGTCGTTGATTTTGATGCTCAGAAGTGTCGTCAG2217
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2442 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION:
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES IN SEQ ID NO:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
ATGGAACTGAAGATTTTGAGTGTCGCGATTGCGACAACATTAACCAGCACTGGCGTATTT60
GCGTTAAGCGAGCCAGTTTCTCAAGTTACAGAGCAACATGC ACATTCGGCTCATACACAC120
GGTGTTGAATTCAATCGAGTTGAATACCAACCAACCGCAACTCTCCCAATTCAGCCCTCT180
AAGGCAACTCGAGTACAGTCACTTGAAAGCCTTGATGAGTCGAGC225
ACTGCTTGTGATTTGG AGGCATTGGTTACCGAAAGCAGTAACCAATTGATCAGCGAAATT285
TTAAGTCAGGGCGCGACGTGTGTGAACCAGTTATTCTCTGCTGAAAGTCGGATTCAAGAG345
TCGGTATTTAGCTCCGATCATATGTACAACATCGCTAAGCACACTACGACGTTGGCGAAG 405
GGGTATACGGGTGGCGGGAGCGATGAACTAGAAACGTTGTTCTTATACTTACGCGCGGGT465
TATTACGCCGAGTTTTACAATGACAACATCTCATTTATTGAATGGGTCACCCCAGCGGTG525
AAAGAATCAGTGGATGCGTTTGTTAACACAGCAA GCTTCTACGAGAACAGCGACCGTCAC585
GGCAAAGTGCTTAGTGAGGTCATCATCACTATGGATAGTGCGGGCTTGCAGCACGCGTAC645
TTACCGCAAGTGACCCAGTGGCTTACTCGTTGGAATGATCAATACGCCCAGCACTGGTAT705
ATGCGCAAT GCGGTTAACGGTGTTTTCACTATTTTGTTTGGTGGGCAGTGGAACGAGCAA765
TTTGTGCAAATAATTGGCAACCAAACGGACCTTGCCAAAGCTTTAGGCGATTTTGCTCTA825
AGGGCGTCATCAATCGGTGCTGAAGATGAGTTTATGGCCGCGAATGCGGGGC GAGAGCTC885
GGGCGTCTGACCAAGTATACGGGTAACGCGAGTTCTGTTGTGAAGAGTCAGCTGAGTCGA945
ATCTTTGAACAGTATGAAATGTATGGTCGGGGTGACGCGGTTTGGCTTGCGGCGGCGGAC1005
ACCGCCTCATATTACGCAGATTGTAGT GAGTTCGGAATTTGTAATTTCGAAACTGAGCTA1065
AAAGGCTTGGTGCTATCGCAAACTTATACTTGTAGCCCGACAATCCGAATTTTGTCTCAG1125
AATATGACGCAAGAGCAACACGCGGCCGCATGTTCTAAAATGGGTTACGAAGAGGGTTAC1185
T TTCATCAGTCATTAGAAACTGGTGAACAGCCAGTAAAAGATGACCACAATACTCAGCTC1245
CAAGTCAATATATTCGATTCAAGTACCGATTATGGTAAGTACGCAGGGCCAATTTTCGAT1305
ATTAGTACTGACAATGGCGGTATGTACTTGGAGGGCGACCCTTCC CAGCCGGGGAATATT1365
CCCAACTTTATTGCTTATGAAGCCTCTTATGCGAACGCAGATCACTTTGTCTGGAACTTA1425
GAGCACGAATACGTGCATTACTTAGATGGTCGATTTGATCTCTATGGAGGGTTTAGTCAT1485
CCAACTGAAAAAATAGTGTG GTGGAGTGAAGGCATTGCAGAGTATGTCGCTCAAGAAAAT1545
GACAACCAAGCAGCACTTGAGACGATTCTAGACGGTTCGACATATACCTTAAGTGAGATT1605
TTCGAGACTACTTATGATGGGTTTGATGTCGATCGAATTTATCGTTGGGGGTACTTAGCT1 665
GTACGTTTTATGTTTGAAAATCATAAAGATGACGTAAACCAAATGCTGGTGGAAACACGC1725
CAAGGGAATTGGATCAATTACAAGGCCACGATCACCCAATGGGCGAATTTGTATCAAAGT1785
GAGTTTGAGCAGTGGCAGCAAACCCTTGTCTCAAATGG TGCTCCTAATGCAGTCATAACC1845
GCAAACAGTAAGGGGAAAGTCGGTGAAAGCATTACATTTAGCAGTGAAAACAGTACAGAC1905
CCAAACGGGAAGATCGTCAGCGTCTTATGGGACTTCGGTGATGGCTCGACAAGTACACAA1965
ACCAAGCCGACG CACCAATATGGGAGTGAAGGGGAGTATTCGGTCAGCCTAAGTGTGACA2025
GACAGTGAAGGCTTGACGGCAACCGCCACTCATACTGTTGTTATCTCAGCGTTGGGCGGT2085
AATGACACATTGCCACAAGACTGCGCGGTGCAAAGTAAAGTAAGCGGTGGGCGCTT AACA2145
GCAGGAGAACCAGTTTGCTTGGCAAATCAACAAACCATTTGGCTGAGCGTACCAGCGGTG2205
AATGAGAGCTCAAACCTGGCGATAACGACGGGGAATGGTACGGGCAACCTAAAGCTTGAA2265
TACAGTAACTCTGGTTGGCCGGATGATACT AATCTTCACGGGTGGTCAGATAATATTGGT2325
AATGGAGAGTGTATTACGTTGTCAAATCAGAGTAACTACTGGGGCTACGTTAAAGTCTCT2385
GGTGACTTTGAGAATGCCGCCATCGTCGTTGATTTTGATGCTCAGAAGTGTCGTCAG2442
Claims
1. An isolated and purified gene encoding a collagenase of Vibrio alginolyticus and having a restriction map as shown in FIG. 1.
2. A recombinant vector comprising the gene of claim 1.
3. An E. coli host cell transformed with a plasmid comprising the gene of claim 1.
4. A process for the production of a collagenase which comprises cultivating the E. coli host cell of claim 3 to express the collagenase, and recovering the collagenase.
5. The gene according to claim 1, which is the 2.7 kb HpaI-EcoRV fragment shown in the restriction map of FIG. 1.
6. A recombinant vector comprising the gene of claim 5.
7. An E. coli host cell transformed with a plasmid comprising the gene of claim 5.
8. A process for the production of a collagenase which comprises cultivating the E. coli cell of claim 7 to express the collagenase, and recovering the collagenase.
9. The gene according to claim 5, wherein said collagenase comprises peptide fragments of the formulas (a) to (t), the peptide fragments of the formulas (a) and (c) to (t) being shown in SEQ ID Nos. 3-21:
- (a): S Q L S R
- (b): I Y R
- (c): Y T G N A S S V V K
- (d): A S S I G A E D E F M A A N A G R E
- (e): E S V D A F V N
- (f): Q G N W I N Y K
- (g): M G Y E E G Y F H Q S L
- (h): A L G D F A L R
- (i): W G Y L A V R
- (j): A G Y Y A E
- (k): V W W S E
- (l): W V T P A V K E
- (m): L D G R F D L Y G G F S H P T E K
- (n): Y N D N I S F
- (o): S S T D Y G K Y A G P I F D
- (p): G D P S Q P G N I P N F I A Y E
- (q): Y V H Y L D G R F D
- (r): T A S Y Y A D C S E
- (s): W N D Q Y
- (t): G Y T G G G S D E L
10. An isolated and purified gene encoding a collagenase having an amino acid sequence comprising the amino acid sequence of the following formula as shown in SEQ ID No. 22: ##STR2## wherein X is hydrogen, and wherein A is alanine, C is cysteine, D is aspartic acid, E is glutamic acid, F is phenylalanine, G is glycine, H is histidine, I is isoleucine, K is lysine, L is leucine, M is methionine, N is asparagine, P is proline, Q is glutamine, R is arginine, S is serine, T is threonine, V is valine, W is tryptophan and Y is tyrosine.
11. An isolated and purified gene encoding a collagenase having an amino acid sequence consisting of the amino acid sequence of the following formula as shown in SEQ ID No. 22: ##STR3## wherein X is hydrogen, and wherein A is alanine, C is cysteine, D is aspartic acid, E is glutamic acid, F is phenylalanine, G is glycine, H is histidine, I is isoleucine, K is lysine, L is leucine, M is methionine, N is asparagine, P is proline, Q is glutamine, R is arginine, S is serine, T is threonine, V is valine, W is tryptophan and Y is tyrosine.
12. A recombinant vector comprising the gene of claim 10 or 11.
13. An E. coli host cell transformed with a plasmid comprising the gene of claim 10 or 11.
14. A process for the production of a collagenase which comprises cultivating the host cells of claim 13 to express the collagenase, and recovering the collagenase.
15. An isolated and purified gene encoding a collagenase precursor having an amino acid sequence comprising the amino acid sequence of the following formula as shown. in SEQ ID No. 23: ##STR4## wherein X is a polypeptide of the formula: ##STR5## and wherein A is alanine, C is cysteine, D is aspartic acid, E is glutamic acid, F is phenylalanine, G is glycine, H is histidine, I is isoleucine, K is lysine, L is leucine, M is methionine, N is asparagine, P is proline, Q is glutamine, R is arginine, S is serine, T is threonine, V is valine, W is tryptophan and Y is tyrosine.
16. An isolated and purified gene encoding a collagenase precursor having an amino acid sequence consisting of the amino acid sequence of the following formula as shown in SEQ ID No. 23: ##STR6## wherein X is a polypeptide of the formula: ##STR7## and wherein A is alanine, C is cysteine, D is aspartic acid, E is glutamic acid, F is phenylalanine, G is glycine, H is histidine, I is isoleucine, K is lysine, L is leucine, M is methionine, N is asparagine, P is proline, Q is glutamine, R is arginine, S is serine, T is threonine, V is valine, W is tryptophan and Y is tryosine.
17. A recombinant vector comprising the gene of claim 15 or 16.
18. An E. coli host cell transformed with a plasmid comprising the gene of claim 15 or 16.
19. A process for the production of a collagenase which comprises cultivating the host cells of claim 18 to express the collagenase, and recovering the collagenase.
20. An isolated and purified gene encoding a collagenase of Vibrio alginolyticus which comprises a DNA sequence of the following formula as shown in SEQ ID No. 24: ##STR8## wherein Z is hydrogen.
21. An isolated and purified gene encoding a collagenase precursor of Vibrio alginolyticus which comprises a DNA sequence of the following formula as shown in SEQ ID No. 25: ##STR9## wherein Z is a DNA sequence of the formula: ##STR10##
22. A process for the production of a collagenase of Vibrio alginolyticus which comprises:
- inserting Bam HI linker into Hpa I site at Base No. 1213 on a DNA fragment of 7 kb composing the plasmid pLCO-1 of Escherichia coli JM 101 (FERM BP-3113) and inserting Sal I linker into Eco RV site at Base No. 3936 on the DNA fragment;
- cleaving said pLCO-1 containing the two linkers with Bam HI and Sal I to obtain a DNA fragment of 2.7 kb containing an entire collagenase gene;
- inserting said DNA fragment into Bam HI/Sal I site of a vector to obtain a recombinant plasmid;
- transforming Escherichia coli with said recombinant plasmid to obtain host cells;
- cultivating said host cells to express the collagenase; and
- recovering the collagenase.
| 4760025 | July 26, 1988 | Estell et al. |
| 4966846 | October 30, 1990 | Deutch et al. |
| 5145681 | September 8, 1992 | Fortney et al. |
| 5177017 | January 5, 1993 | Lin et al. |
| 115974 | August 1984 | EPX |
| 309879 | April 1989 | EPX |
- Biochimica Et Biophysica Acta, vol. 874, No. 3, 1986, pp. 296-304. Gene, vol. 76, 1989, pp. 281-288. Biochimica Et Biophysica Acta, vol. 522, No. 1, 1978, pp. 218-222. Chemical Abstracts, vol. 85, 1976, p. 216, abstract No. 58688b. Hare, P., et al., 1983, Journal of General Microbiology, 129:1141-1147. Young, R. A., et al., 1983, Science, 222:778-782. Taylor, M. J., et al., 1979, Advances in Applied Microbiology, 25:7-35. Fukushima, J., et al., 1989, Journal of Bacteriology, 171(3):1698-1704. Atsumi, Y., et al., 1989, Journal of Bacteriology 171(9): 5173-5175. Pharmacia Cabalogne, 1986, p. 60.
Type: Grant
Filed: Nov 27, 1990
Date of Patent: Sep 26, 1995
Assignee: Suntory Limited (Osaka)
Inventors: Yuji Shibano (Toyonaka), Kazuyuki Morihara (Shimonoseki), Kenji Okuda (Yokohama), Jun Fukushima (Tokyo)
Primary Examiner: Robert A. Wax
Assistant Examiner: William W. Moore
Law Firm: Wenderoth, Lind & Ponack
Application Number: 7/618,946
International Classification: C12N 952; C12N 1557; C12N 1570;
